Heretofore, a trehalose phosphorylase derived from Euglena gracilis has been reported as EC 2.4.1.64 in Enzyme Nomenclature 1992 (Academic Press). Also, an enzyme derived from Flammulina velutipes which produces trehalose from .alpha.-glucose 1-phosphate and glucose has been reported as a trehalose phosphorylase (FEMS Microbiology Letters, 55, 147-150 (1988)).
However, the above mentioned trehalose phosphorylase derived from Flammulina velutipes has not sufficiently been purified due to its low stability. Therefore, no sufficient enzymic properties thereof have been known, for example, pH and temperature stability, optimal reaction temperature or molecular weight, furthermore, no sufficient data for the optimal reaction pH or substrate specificity has been reported. In addition, its productivity of trehalose from .alpha.-glucose 1-phosphate and glucose was unsatisfactory.
The trehalose phosphorylase disclosed as EC 2.4.1.64 in Enzyme Nomenclature 1992 (Academic Press) is a trehalose phosphorylase which produce trehalose from .beta.-D-glucose 1-phosphate and D-glucose.
The inventors have been investigating to find a novel thermally stable trehalose phosphorylase which produces trehalose from .alpha.-D-glucose 1-phosphate and D-glucose and found that microorganisms of Grifola, Pleurotus, Lyophyllum, Lentinus, Agaricus, Trametes, Coriolus, Lenzites, Schizophyllum, Panus, Crepidotus, Laetiporus, Polyporellus, Favolus, Trichaptum, Oudemansiella, Naematoloma, Rhodophyllus, Gloeophyllum, Fomes, Ganoderma, Elfvingia, Fomitopsis, Armillariella, Lampteromyces, Pholiota, and Tricholoma genera can produce trehalose phosphorylases. The inventors also found that these trehalose phosphorylases obtained from the above mentioned cultured microorganisms are thermally stable and accomplished the present invention.
This invention is based on the findings of enzymes which produce trehalose from .alpha.-D-glucose 1-phosphate and D-glucose. The trehalose phosphorylases of the present invention exhibit clearly different substrate specificity from that of the known enzymes producing trehalose from .beta.-D-glucose 1-phosphate and D-glucose shown above. The trehalose phosphorylases of the present invention can catalyze the reaction of .alpha.-D-glucose 1-phosphate with D-glucose to produce trehalose.
The thermally stable trehalose phosphorylases can be obtained as purified single enzymes for the first time by the present invention and their physicochemical properties were elucidated.